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Insecticide sensitivity of native chloride and sodium channels in a mosquito cell line
Pesticide Biochemistry and Physiology 130 (2016) 59–64
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Insecticide sensitivity of native chloride and sodium channels in a mosquito cell line Lacey J. Jenson a,b, Troy D. Anderson b, Jeffrey R. Bloomquist a,⁎ a b
Emerging Pathogens Institute, Department of Entomology and Nematology, University of Florida, Gainesville, FL 32601, United States Virginia Polytechnic Institute and State University, Department of Entomology, Blacksburg, VA 24061, United States
a r t i c l e
i n f o
Article history: Received 16 June 2015 Received in revised form 25 November 2015 Accepted 27 November 2015 Available online 28 November 2015 Keywords: DIDS Fenvalerate Lindane Sua1B Tetrodotoxin Veratridine
a b s t r a c t The aim of this study was to investigate the utility of cultured Anopheles gambiae Sua1B cells for insecticide screening applications without genetic engineering or other treatments. Sua1B cells were exposed to the known insecticidal compounds lindane and DIDS, which inhibited cell growth at micromolar concentrations. In patch clamp studies, DIDS produced partial inhibition (69%) of chloride current amplitudes, and an IC50 of 5.1 μM was determined for Sua1B cells. A sub-set of chloride currents showed no response to DIDS; however, inhibition (64%) of these currents was achieved using a low chloride saline solution, confirming their identity as chloride channels. In contrast, lindane increased chloride current amplitude (EC50 = 116 nM), which was reversed when cells were bathed in calcium-free extracellular solution. Voltage-sensitive chloride channels were also inhibited by the presence of fenvalerate, a type 2 pyrethroid, but not significantly blocked by type 1 allethrin, an effect not previously shown in insects. Although no evidence of fast inward currents typical of sodium channels was observed, studies with fenvalerate in combination with veratridine, a sodium channel activator, revealed complete inhibition of cell growth that was best fit by a two-site binding model. The high potency effect was completely inhibited in the presence of tetrodotoxin, a specific sodium channel blocker, suggesting the presence of some type of sodium channel. Thus, Sua1B cells express native insect ion channels with potential utility for insecticide screening. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Due to the increasing resistance demonstrated by insects to pyrethroids, especially disease vectors [1], the need for insecticides with novel modes of action is becoming increasingly important. Development of new insecticides is also vital because regulations and use restrictions on conventional insecticides are becoming more stringent [2]. Invention of new insecticide chemical classes is limited by available chemical synthesis technologies, as well as the necessary procurement of large amounts of target site protein needed for high throughput testing. Smagghe et al. [3] reviewed the use of insect cell lines as tools in virus-related research, as models in the study
Abbreviations: CACC, calcium-activated chloride channel; CI, 95% confidence intervals; DIDS, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid; EC50, effective concentration at half maximal effect; IC50, inhibitory concentration at 50% of initial effect; FNV, fenvalerate; GABA, gamma-aminobutyric acid; TTX, tetrodotoxin; VSCC, voltage-sensitive chloride channel; VTD, veratridine. ⁎ Corresponding author at: Emerging Pathogens Institute, Department of Entomology and Nematology, 2055 Mowry Road, PO Box 100009, University of Florida, Gainesville, FL 32601, United States. E-mail address: [email protected]fl.edu (J.R. Bloomquist).
http://dx.doi.org/10.1016/j.pestbp.2015.11.012 0048-3575/© 2015 Elsevier Inc. All rights reserved.
of signal mechanisms and gene expression, and as platforms for screening novel insecticide activities, receptors, and ligands for pest control. When used for insecticide screening, cell lines typically contain the cloned gene of interest, coupled to a fluorescent reporter system [4]. However, there is a relative paucity of information available on the subunit composition of insect ion channels and receptors compared to their mammalian homologs, and they can sometimes be difficult to express in cell lines. The main goal of this research was to assess any expression of insecticide target sites, especially Cl− and Na+ channels from an undifferentiated (native) mosquito cell line, in order to avoid cloning/expression issues. Previous screens of established insecticides on cell growth of an undifferentiated Spodoptera exigua cell line found it was sensitive to mitochondrial-directed compounds, but not insect growth regulators or agents acting specifically upon neuronal targets [5]. The present study used established pharmacological probes, cell growth, and patch clamp techniques, to detect expression of targets in Sua1B cells, a cell line first isolated from neonate Anopheles gambiae larvae [6]. Screens focused mostly on compounds affecting VSCCs, since these are known to exist in Sua1B cells [7], and blockers of these channels are insecticidal [8]. Special attention was made to correlate ion channel effects with changes in cell growth. The results could lead to new insecticides and/ or high throughput screening methods.
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2. Materials and methods 2.1. Chemicals Lindane, DIDS, VTD, and FNV were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) and were dissolved in DMSO (Fisher Scientific, Pittsburgh, PA, USA). TTX was obtained from Abcam (Cambridge, MA, USA), was dissolved in deionized water and stored as frozen 1 mM aliquots at −80 °C before use. 2.2. General cell culture techniques The Sua1B insect cell line was obtained from Dr. Michael Povelones of Professor Fotis Kafatos' laboratory (Imperial College, London, UK). The cells were derived from triturated A. gambiae neonate larvae and maintained as described previously [6]. Cells were maintained in a log phase culture in tissue culture flasks (BD Falcon, Fisher Scientific, Suwanee, Georgia, USA) treated with Schneider's insect media from Sigma-Aldrich (St. Louis, Missouri, USA). Insect media was supplemented with 10% fetal bovine serum (Sigma) and 100 U/ml penicillin and streptomycin (Sigma). Cells were maintained at 28 °C in a non-humidified environment without CO2 amendment (Amerex Instruments Inc., Lafayette, CA). Cells were passaged every 3–5 days. 2.3. Treatment of cells with insecticides Once a confluent monolayer of cells formed in the culture flask, the cells were sloughed off and transferred to another sterile flask. The quantity of cells transferred was determined by the area of the growth surface to give a 1:5 dilution (cells/cm2), as in a normal passage. Fresh growth medium was added and the cells were allowed to attach for 30 min in the incubator. The media was then removed and fresh media containing either 0.1% DMSO (control) or test compound in vehicle was added to the culture. 2.4. Cell counting For cell number experiments, counts were performed using the methods of Jenson et al. [9]. Briefly, an ocular grid (0.221 mm2) was aligned with the corners of the scored lines on the flask so that the same places and area were counted each day. The number of cells and the number differentiated within the grid were recorded visually every day for 3 days. Two counts were taken in areas at each of four intersections so that 8 samples were taken per flask. Each experiment used the same experimental design with at least three replicate flasks. 2.5. Manual whole cell patch clamp and chemical application Pyrethroid effects on chloride currents were made by manual patch clamp. Currents were measured with an Axopatch 200B patch clamp amplifier (Molecular Devices, Sunnyvale, VA, USA), connected via A/Dconverter (Digidata 1440A, Molecular Devices) to a personal computer. Cells were maintained at a holding potential of −100 mV, and stepped up to +100 mV in 10 mV increments and 500 msec duration. Recordings underwent low-pass filtering at 2 kHz, corrected for series resistance, and were sampled at 10 kHz. For recording and analysis, pClamp 10.0 software was used. Cells were bathed in an external buffer of the following composition: 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM d-glucose monohydrate, 10 mM HEPES, pH 7.4, osmolarity: 298 mOsmol. Patch pipettes (0.3 μm tip) were filled with solutions containing 10 mM NaCl, 140 mM KF, 2 mM MgCl2, 20 mM EGTA, 10 mM HEPES, pH 7.2, osmolarity: 288 mOsmol. Solutions of insecticide or drug were prepared in DMSO and diluted for patch clamping experiments into extracellular buffer to the corresponding concentrations with no more than 1% DMSO. After establishing a whole-cell clamp, the cell was perfused with drug via a gravity
driven perfusion system. Fluid was controlled with pinch valves releasing fluid from the reservoir into the drug ejection pipette (0.83 mm) for microperfusion over the target cell. 2.6. Planar patch clamp Planar patch clamp was used for all other electrophysiological experiments. Confluency of cells when harvested for planar patching was between 50%-80%. Cells were washed with 1 × PBS (Ca2 + and Mg2+-free). Schneider's insect media (9 mL) containing 10% fetal bovine serum (Sigma) and 100 U/ml penicillin and streptomycin (Sigma) was added to the cells and the flask was scraped. Dislodged cells were then centrifuged for 2 min at 100 g and supernatant was discarded. Cells were then re-suspended in external recording solution at a final density of 1 × 106–5 × 107 cells/ml (200–500 μL). Cells were pipetted onto a glass chip whose base was under negative pressure via Port-a-Patch® system (Nanion Technologies Inc., North Brunswick, NJ, USA). After sealing and membrane rupture to establish whole cell clamp, currents were amplified and filtered by a patch-clamp amplifier (HEKA EPC 10), connected to a personal computer. Cells were maintained at a holding potential of −80 mV, and stepped up to +100 mV in 10 mV increments. Steps were for 500 msec duration. Recordings underwent low-pass filtering at 2 kHz and were sampled at 10 kHz. For recording and data analysis, PatchMaster v2x60 software was used (HEKA Instruments, Bellmore, NY, USA). Chips (2–3.5 MΩ) were prepared with internal buffer of the following composition: 50 mM KCl, 10 mM NaCl, 60 mM KF, 2 mM MgCl2, 20 mM EGTA, 10 mM HEPES, pH 7.2., osmolarity: 288 mOsmol. Cells were prepared and patch clamped in an external buffer of the following composition: 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D-glucose monohydrate, 10 mM HEPES, pH 7.4, osmolarity: 298 mOsmol. 2.7. Statistical analyses The growth of cells was determined with single chemical treatment or multiple agents in combination, over time. Cell number over five days was analyzed for each treatment alone or in combination. The mean and SEM of treatment data points were then calculated. Treatment means were compared by using a one-way analysis of variance procedure (ANOVA), and a Student–Newman–Keul's multiple comparison test would follow if a significant treatment effect was observed. Concentration-response curves were constructed from growth measurements or voltage clamp currents, the latter taken at maximal current amplitude during a 500 msec activating pulse. Curves were generated by non-linear regression of log[agonist] vs. response or to a two-site binding model with GraphPad Prism™ (GraphPad Software, San Diego, CA, USA). Data were analyzed for each curve to determine the IC50 or EC50, 95% CI, and R2 (goodness of fit), as well as maximal effect of the treatment. 3. Results The known chloride channel blockers lindane and DIDS were tested against Sua1B cells to characterize any effects on cell growth (Fig. 1). A time-dependent response was demonstrated for Sua1B cells in the presence of varying concentrations of lindane (0.5–200 μM). No significant inhibition of cell number was observed on day 1 (Fig. 1A). Sua1B cell numbers were significantly inhibited at high concentrations of lindane, with 200 μM showing the highest amount of inhibition on days 2 (73%) and 3 (82%), with less inhibition of cell growth as concentrations decreased to 50 μM. No statistical significance was found in the response to lindane for lower concentrations (0.5–10 μM) on days 1–3 when compared to control (Fig. 1A). A concentration-response plot for lindane was run on Sua1B cell growth over a period of 3 days (Fig. 1B). Inhibition of cell growth after 1 day of exposure was modest and an IC50 value could not be reliably determined. The IC50 (CI) value for day 2
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Fig. 1. Effects of the organochlorine insecticide lindane on Sua1B insect cell number. (A) Time course curves for varying concentrations of lindane on Sua1B cells. (B) Concentrationresponse curves of lindane effects on Sua1B cell number over culture days 1–3. In B, the numbers of cells in solvent-treated controls are identical to those observed at the lowest concentration of lindane. In both plots, symbols are means ± SEM, and when error bars are absent, they are within the size of the symbol.
was 87 (44–172) μM, R2 = 0.84, and Hill slope of −0.67; and for day 3 was 68 (34–135) μM, R2 = 0.85, and a Hill slope = −0.72 (Fig. 1B). Sua1B cells displayed an increased Cl− current amplitude after the application of lindane (Fig. 2A,B), whereas DMSO (0.1%) showed no change. The effect of lindane varied with concentration, and was elevated significantly above basal levels at 100 μM (t-test, P b 0.05). Lindane stimulated chloride currents to a calculated maximum of 144% of control, with an EC50 of 116 (21–658) nM, R2 = 0.23, and a Hill slope of 1.6 (Fig. 2C). Lindane enhancement of chloride currents was blocked in extracellular solution without calcium ions (Fig. 2C). Current amplitudes in 1 mM lindane were lower than expected, and the current amplitude declined both with and without calcium. This effect may result from chloride current block by lindane at this concentration, and these data distorted the plot and curve fit, as reflected in the low R2 value (0.23). DIDS, a VSCC blocker [10], showed significant inhibition of cell growth at high concentrations, with 100 μM active on day 1 and declining in effect thereafter, while 200 μM was effective on all 3 days of incubation, when compared to control (Fig. 3A). No significant differences were observed on cell growth in the presence of 50 μM DIDS. A concentrationresponse plot for DIDS was run on Sua1B cell growth at day 2 of exposure, which showed an IC50 = 53 (48–80) μM, R2 = 0.93, and a Hill slope = 0.75 (Fig. 3B). The calculated minimum for the curve was 439 cells, for a maximal 46% inhibition of cell growth by DIDS. Because DIDS is a known blocker of Cl− currents and reduced the number of Sua1B cells, its effects on Cl− currents were studied using whole cell planar patch electrophysiological techniques. Cells displayed reduced current amplitude after the application of DIDS (100 nM1 mM), especially at positive potentials, and with increased inhibition at higher concentrations (10 μM-1 mM) (Fig. 4A). DMSO (0.1%)
controls showed no effect on the amplitude of the chloride current. A concentration-response plot for DIDS was run on Sua1B cells on day 2 of exposure, which showed an IC50 = 5.1 μM (0.15–174.5 μM), a Hill slope = −0.5, and an R2 = 0.63 (Fig. 4B). Sua1B cells only showed partial inhibition of Cl− current (69%) with DIDS even at high concentrations (1 mM). Some chloride currents (7 out of 19 patches) did not respond to DIDS inhibition, so recordings were performed in a low chloride buffer to confirm that they were chloride currents (Fig. 5A,B). In the presence of low chloride solution (35 mOsmol Cl− in low chloride saline solution compared to 297 mOsmol Cl− in regular external saline solution), DIDSinsensitive currents showed significantly reduced current amplitude, confirming that they were indeed chloride currents (Fig. 5A,B). No early inward currents consistent with fast sodium channels were observed in patch clamp studies. Chloride currents were also exposed to a pyrethroid of both the type 1 (allethrin) and type 2 (FNV) categories, respectively [11,12], and compared to control currents in DMSO (0.1%) to determine any significant effects on overall current amplitudes (Fig. 6). Control Cl− currents (Fig. 6A) were reduced about 27% by 10 μM allethrin (Fig. 6B), but substantially reduced by this same concentration of FNV (Fig. 6C). Replicated current–voltage relationships of different cells (n = 6) showed no significant reduction of current amplitudes in Sua1B cell recordings after application of 10 μM allethrin (Fig. 6D) when compared to control Cl− current amplitudes. In contrast, FNV (10 μM) displayed 60–70% reduction in current amplitude at this concentration when compared to control currents at −100 and +100 mV (Fig. 6E). In a previous study [13], we found that when applied to Sua1B cells alone, 1 μM VTD (a sodium channel activator) and TTX (a sodium
Fig. 2. Illustration of chloride channel currents under control (A) conditions and in the presence of 1 mM lindane (B). C. Concentration-dependence of increased chloride current in Sua1B cells when exposed to lindane (n = 9); extracellular solution containing calcium compared to the effect of lindane applied in extracellular solution without calcium (n = 6). Symbols are means ± SEM bars. Asterisks indicate a significant difference in lindane effects at a given concentration, in the presence and absence of calcium (unpaired T-test, P b 0.05).
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Fig. 3. Effects of DIDS on the number of Sua1B cells (A) at various incubation times (n = 3). Bars are means ± SEM. Statistical analysis was performed for each post-treatment day using one-way ANOVA (P b 0.05) and a Dunnett's multiple comparison post test. Letters represent statistical difference for a given day of exposure, and bars labeled by different letters are significantly different. Concentration-response curve of DIDS (B) on the number of Sua1B cells, corresponding to data for day 2 of the incubation (n = 3). Symbols are means ± SEM bars. When error bars are absent, they are within the size of the symbol.
channel blocker) showed no effect on cell growth. In the present investigation, when VTD was applied in combination with varying amounts of FNV, there was inhibition in cell number that was fit by a two-site binding model (Fig. 7). A potent effect of IC50 = 15 nM (4.5–49 nM), was found for the high affinity site and a lower potency effect with IC50 = 56 μM (25–124 μM) was found for the second site. The biphasic curve fit had an R2 = 0.951. Sua1B cells were then exposed to TTX over a 3 day period with combinations of VTD (1 μM) + FNV (1 μM or 100 μM). In these experiments, 1 μM TTX reversed the inhibitory effect of 0.1 μM FNV + 1 μM VTD on cell number, whereas high concentrations of FNV (100 μM + 1 μM VTD) showed inhibition of cell numbers that could not be reversed by TTX (Fig. 7, inset). 4. Discussion Two types of chloride channels previously identified in Sua1B cells [7] were characterized within this study. Outwardly rectifying voltage sensitive currents observed in native Sua1B cells seem to be mediated either primarily by CACCs or by a combination of CACCs and volumesensitive, outwardly-rectifying anion channels [7]. High concentrations of lindane, an organochlorine insecticide, inhibited growth of Sua1B cells, but the response did not occur in a typical concentration-dependent manner. Current amplitudes were increased at concentrations below 1 mM, which then reduced current amplitude (Fig. 2). The primary neurotoxic effects of lindane are usually attributed to its interference with γ-aminobutyric acid (GABA) receptor-ionophore complex; however, Abalis et al. [14] showed that lindane also binds with high affinity (Ki = 0.04 ± 0.01 μM) to VSCC in Torpedo electroplaque. Lindane, at the concentrations run in this study (50–200 μM), also affects intracellular calcium mobilization in rat myometrial smooth muscle cells (100–200 μM) [15], guinea pig macrophages (125 μM) [16] and
human HL-60 cells [17]. Such calcium mobilization could explain the effects of lindane at high concentrations on Sua1B cell growth, as well as activation of CACC (Fig. 3). However, any lindane-specific alterations in calcium ion homeostasis or signaling remain to be determined. Future studies utilizing calcium fluorescence assays in the presence and absence of lindane should be run to verify any possible effects on calcium ion flux/homeostasis. Outward Cl− currents expressed in Sua1B cells were mostly blocked by DIDS. The insecticidal/nematicidal properties of DIDS have been documented by Boina et al. [18,19], who observed paralysis in free living and plant parasitic nematodes, and was a stomach poison in European corn borer (Ostrinia nubilalis) larvae. Additional experiments on European corn borer disclosed that DIDS had a dose-dependent effect on larval midgut 36Cl− ion transport (IC50 = 7.4 μM) and midgut alkalinity (ca. IC50 = 29 μM) [19]. This latter action was similar to the potency found for DIDS on cell growth in this study (IC50 = 53 μM). Using the planar patch clamp technique, the inhibitory effect of DIDS on Cl− currents at +60 mV had IC50 = 5.1 μM, which is 10-fold greater potency than that observed in the cell growth studies, but similar to the IC50 for blocking 36Cl− uptake in European corn borer larval midgut. A similar potency of DIDS on Cl− currents was shown in rat peritoneal mast cells with IC50 = 2.3 μM and a Hill coefficient n = 0.7 [20]. Not all cells showed inhibition of current amplitude by DIDS, and this quality is shared by the cystic fibrosis transmembrane Cl− conductance regulator [21] and a certain ClC-3 type of chloride channel expressed in CHO cells [22]. The identity of the DIDS insensitive Cl− channel of Sua1B cells and the amino acids responsible for DIDS sensitivity in these channels remain to be determined. When characterizing the chloride channels present in Sua1B cells, this study found the majority of channels to be VSCCs, while those Diykov et al. [7] found were predominantly the CACC channel subtype.
Fig. 4. Electrophysiological effects of DIDS on chloride currents in Sua1B cells. (A) Current–voltage relationship of chloride current in Sua1B cells and inhibition with DIDS (n = 6). Symbols are means with error bars omitted for clarity. (B) Concentration-response curve representing percent inhibition of DIDS at +60 mV (n = 6). Symbols are means ± SEM bars.
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Fig. 5. DIDS insensitive chloride currents under planar patch conditions. Traces in (A) and (B) illustrate currents in normal (297 mOsmol) and low (35 mOsmol) chloride extracellular solutions, respectively. Plot (C) shows current–voltage relationship of DIDS-insensitive chloride channels in control and low chloride solutions (n = 3). Symbols are means ± SEM bars. Asterisks indicate a significant difference in current amplitude at a given voltage step in control or low chloride conditions (unpaired T-test, P b 0.05).
Both chloride channel subtypes were found in each study, but are thought to activate in different physiological circumstances and occur independently of one another. We do not know why the variation in chloride channel expression occurs, but it could be due to different aliquots/passages of Sua1B cells. Both studies showed the same reduction in current amplitude in the presence of DIDS (69%), confirming that a similar population of DIDS-sensitive chloride channels was present. Patch clamped Sua1B cells lack inward currents indicative of typical electrogenic sodium or calcium channels. However, in the presence of 1 μM VTD and increasing concentrations of FNV, cell death was increased in a dose-dependent, biphasic manner (Fig. 7). The high potency effect (EC50 = 15 nM) was completely inhibited in the presence of TTX, suggesting the possible presence of an “electrically-silent” sodium channel, similar to that previously described in rat C6 [23] and C9 cells [24]. In these cells, sodium currents cannot be activated by voltage clamp depolarization, but radiotracers can be used to measure chemical
activation of the channels by alkaloid or protein neurotoxins. Similar experiments could be used to characterize these putative sodium channels in Sua1B cells. The signs of intoxication by pyrethroids differ between the two categories (type 1 and 2) in mammals, where compounds described as type 1 (e.g., allethrin) display a tremor syndrome, whereas type 2 (e.g., FNV) present choreoathetosis (sinuous writhing) and salivation [25]. The different intoxication syndromes of the two classes of pyrethroid led to the hypothesis that there are other mechanisms involved in the toxicity of pyrethroids than just an action on sodium channels. Relevant to the present study is a proposed effect on voltage-gated chloride channels. Forshaw and Ray [26] showed a reduction of chloride conductance and an increase in membrane resistance in mammalian skeletal muscle, non-myelinated nerve fibers, and mouse neuroblastoma cells in the presence of a type 2 pyrethroid (deltamethrin), but not to a type 1 (cismethrin). In Sua1B cells, we observed an inhibition of
Fig. 6. Effects of pyrethroids on Sua1B cells using manual whole cell patch clamping technique. (A) shows control chloride current of a Sua1B cell before treatment. (B) and (C) are examples of chloride current inhibition by type 1 (allethrin) and type 2 (FNV) pyrethroids, respectively (10 μM each). Panels (D) and (E) show current–voltage relationships of control chloride currents and those currents in the presence of type 1 allethrin (n = 6, 10 μM) and type 2 FNV (n = 4, 10 μM) class of pyrethroids. Symbols are means + SEM and asterisks indicate a significant difference in current amplitude at a given voltage step (unpaired T-test, P b 0.05).
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[5]
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[9]
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[12] Fig. 7. Inhibition of cell growth by FNV in the presence of a constant 1 μM concentration of VTD on cell number in Sua1B cells, and block of this effect by 1 μM TTX. Concentrationresponse curve of type 2 pyrethroid FNV taken on day 3 of incubation (n = 3 replicate experiments run on different days with different flasks of cells) is fit by a two site model. Symbols are means ± SEM bars. When error bars are absent, they are within the size of the symbol. Inset: TTX sensitivity of high potency (10−7 M FNV) and low potency (10−4 M FNV) effects, in separate experiments with matched controls (n = 3). Statistical analysis was performed using one-way ANOVA (P b 0.05) and a Student–Newman–Keul's multiple comparison post test. Bars labeled by different letters represents statistical significance (P b 0.05).
[13]
[14]
[15]
[16]
chloride current amplitude after application of 10 μM FNV (type 2), while no significant inhibition of the chloride current was shown in the presence of 10 μM allethrin (type 1). Such an effect of FNV on VSCC could contribute to the low potency inhibitory effect of FNV on Sua1B cell growth, but could also be due to mitochondrial effects, since Gassner et al. [27] showed pyrethroids are micromolar inhibitors of mitochondrial complex 1 in rat hepatocytes. To our knowledge, this finding is the first report of type 2 pyrethroid inhibition of insect VSCCs, and further research is required to explore any role it plays in pyrethroid intoxication and lethality.
[17]
Acknowledgments
[22]
This work was supported by USDA Specific Cooperative Agreement 58-0208-0-068 (to JRB) as part of the Deployed War Fighter Research Program. We would like to extend a special thanks to Dr. Michael Povelones for providing us with a stock of the Sua-1B cells.
[23]
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Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Insecticide sensitivity of native chloride and sodium channels in a mosquito cell line Lacey J. Jenson a,b, Troy D. Anderson b, Jeffrey R. Bloomquist a,⁎ a b
Emerging Pathogens Institute, Department of Entomology and Nematology, University of Florida, Gainesville, FL 32601, United States Virginia Polytechnic Institute and State University, Department of Entomology, Blacksburg, VA 24061, United States
a r t i c l e
i n f o
Article history: Received 16 June 2015 Received in revised form 25 November 2015 Accepted 27 November 2015 Available online 28 November 2015 Keywords: DIDS Fenvalerate Lindane Sua1B Tetrodotoxin Veratridine
a b s t r a c t The aim of this study was to investigate the utility of cultured Anopheles gambiae Sua1B cells for insecticide screening applications without genetic engineering or other treatments. Sua1B cells were exposed to the known insecticidal compounds lindane and DIDS, which inhibited cell growth at micromolar concentrations. In patch clamp studies, DIDS produced partial inhibition (69%) of chloride current amplitudes, and an IC50 of 5.1 μM was determined for Sua1B cells. A sub-set of chloride currents showed no response to DIDS; however, inhibition (64%) of these currents was achieved using a low chloride saline solution, confirming their identity as chloride channels. In contrast, lindane increased chloride current amplitude (EC50 = 116 nM), which was reversed when cells were bathed in calcium-free extracellular solution. Voltage-sensitive chloride channels were also inhibited by the presence of fenvalerate, a type 2 pyrethroid, but not significantly blocked by type 1 allethrin, an effect not previously shown in insects. Although no evidence of fast inward currents typical of sodium channels was observed, studies with fenvalerate in combination with veratridine, a sodium channel activator, revealed complete inhibition of cell growth that was best fit by a two-site binding model. The high potency effect was completely inhibited in the presence of tetrodotoxin, a specific sodium channel blocker, suggesting the presence of some type of sodium channel. Thus, Sua1B cells express native insect ion channels with potential utility for insecticide screening. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Due to the increasing resistance demonstrated by insects to pyrethroids, especially disease vectors [1], the need for insecticides with novel modes of action is becoming increasingly important. Development of new insecticides is also vital because regulations and use restrictions on conventional insecticides are becoming more stringent [2]. Invention of new insecticide chemical classes is limited by available chemical synthesis technologies, as well as the necessary procurement of large amounts of target site protein needed for high throughput testing. Smagghe et al. [3] reviewed the use of insect cell lines as tools in virus-related research, as models in the study
Abbreviations: CACC, calcium-activated chloride channel; CI, 95% confidence intervals; DIDS, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid; EC50, effective concentration at half maximal effect; IC50, inhibitory concentration at 50% of initial effect; FNV, fenvalerate; GABA, gamma-aminobutyric acid; TTX, tetrodotoxin; VSCC, voltage-sensitive chloride channel; VTD, veratridine. ⁎ Corresponding author at: Emerging Pathogens Institute, Department of Entomology and Nematology, 2055 Mowry Road, PO Box 100009, University of Florida, Gainesville, FL 32601, United States. E-mail address: [email protected]fl.edu (J.R. Bloomquist).
http://dx.doi.org/10.1016/j.pestbp.2015.11.012 0048-3575/© 2015 Elsevier Inc. All rights reserved.
of signal mechanisms and gene expression, and as platforms for screening novel insecticide activities, receptors, and ligands for pest control. When used for insecticide screening, cell lines typically contain the cloned gene of interest, coupled to a fluorescent reporter system [4]. However, there is a relative paucity of information available on the subunit composition of insect ion channels and receptors compared to their mammalian homologs, and they can sometimes be difficult to express in cell lines. The main goal of this research was to assess any expression of insecticide target sites, especially Cl− and Na+ channels from an undifferentiated (native) mosquito cell line, in order to avoid cloning/expression issues. Previous screens of established insecticides on cell growth of an undifferentiated Spodoptera exigua cell line found it was sensitive to mitochondrial-directed compounds, but not insect growth regulators or agents acting specifically upon neuronal targets [5]. The present study used established pharmacological probes, cell growth, and patch clamp techniques, to detect expression of targets in Sua1B cells, a cell line first isolated from neonate Anopheles gambiae larvae [6]. Screens focused mostly on compounds affecting VSCCs, since these are known to exist in Sua1B cells [7], and blockers of these channels are insecticidal [8]. Special attention was made to correlate ion channel effects with changes in cell growth. The results could lead to new insecticides and/ or high throughput screening methods.
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2. Materials and methods 2.1. Chemicals Lindane, DIDS, VTD, and FNV were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) and were dissolved in DMSO (Fisher Scientific, Pittsburgh, PA, USA). TTX was obtained from Abcam (Cambridge, MA, USA), was dissolved in deionized water and stored as frozen 1 mM aliquots at −80 °C before use. 2.2. General cell culture techniques The Sua1B insect cell line was obtained from Dr. Michael Povelones of Professor Fotis Kafatos' laboratory (Imperial College, London, UK). The cells were derived from triturated A. gambiae neonate larvae and maintained as described previously [6]. Cells were maintained in a log phase culture in tissue culture flasks (BD Falcon, Fisher Scientific, Suwanee, Georgia, USA) treated with Schneider's insect media from Sigma-Aldrich (St. Louis, Missouri, USA). Insect media was supplemented with 10% fetal bovine serum (Sigma) and 100 U/ml penicillin and streptomycin (Sigma). Cells were maintained at 28 °C in a non-humidified environment without CO2 amendment (Amerex Instruments Inc., Lafayette, CA). Cells were passaged every 3–5 days. 2.3. Treatment of cells with insecticides Once a confluent monolayer of cells formed in the culture flask, the cells were sloughed off and transferred to another sterile flask. The quantity of cells transferred was determined by the area of the growth surface to give a 1:5 dilution (cells/cm2), as in a normal passage. Fresh growth medium was added and the cells were allowed to attach for 30 min in the incubator. The media was then removed and fresh media containing either 0.1% DMSO (control) or test compound in vehicle was added to the culture. 2.4. Cell counting For cell number experiments, counts were performed using the methods of Jenson et al. [9]. Briefly, an ocular grid (0.221 mm2) was aligned with the corners of the scored lines on the flask so that the same places and area were counted each day. The number of cells and the number differentiated within the grid were recorded visually every day for 3 days. Two counts were taken in areas at each of four intersections so that 8 samples were taken per flask. Each experiment used the same experimental design with at least three replicate flasks. 2.5. Manual whole cell patch clamp and chemical application Pyrethroid effects on chloride currents were made by manual patch clamp. Currents were measured with an Axopatch 200B patch clamp amplifier (Molecular Devices, Sunnyvale, VA, USA), connected via A/Dconverter (Digidata 1440A, Molecular Devices) to a personal computer. Cells were maintained at a holding potential of −100 mV, and stepped up to +100 mV in 10 mV increments and 500 msec duration. Recordings underwent low-pass filtering at 2 kHz, corrected for series resistance, and were sampled at 10 kHz. For recording and analysis, pClamp 10.0 software was used. Cells were bathed in an external buffer of the following composition: 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM d-glucose monohydrate, 10 mM HEPES, pH 7.4, osmolarity: 298 mOsmol. Patch pipettes (0.3 μm tip) were filled with solutions containing 10 mM NaCl, 140 mM KF, 2 mM MgCl2, 20 mM EGTA, 10 mM HEPES, pH 7.2, osmolarity: 288 mOsmol. Solutions of insecticide or drug were prepared in DMSO and diluted for patch clamping experiments into extracellular buffer to the corresponding concentrations with no more than 1% DMSO. After establishing a whole-cell clamp, the cell was perfused with drug via a gravity
driven perfusion system. Fluid was controlled with pinch valves releasing fluid from the reservoir into the drug ejection pipette (0.83 mm) for microperfusion over the target cell. 2.6. Planar patch clamp Planar patch clamp was used for all other electrophysiological experiments. Confluency of cells when harvested for planar patching was between 50%-80%. Cells were washed with 1 × PBS (Ca2 + and Mg2+-free). Schneider's insect media (9 mL) containing 10% fetal bovine serum (Sigma) and 100 U/ml penicillin and streptomycin (Sigma) was added to the cells and the flask was scraped. Dislodged cells were then centrifuged for 2 min at 100 g and supernatant was discarded. Cells were then re-suspended in external recording solution at a final density of 1 × 106–5 × 107 cells/ml (200–500 μL). Cells were pipetted onto a glass chip whose base was under negative pressure via Port-a-Patch® system (Nanion Technologies Inc., North Brunswick, NJ, USA). After sealing and membrane rupture to establish whole cell clamp, currents were amplified and filtered by a patch-clamp amplifier (HEKA EPC 10), connected to a personal computer. Cells were maintained at a holding potential of −80 mV, and stepped up to +100 mV in 10 mV increments. Steps were for 500 msec duration. Recordings underwent low-pass filtering at 2 kHz and were sampled at 10 kHz. For recording and data analysis, PatchMaster v2x60 software was used (HEKA Instruments, Bellmore, NY, USA). Chips (2–3.5 MΩ) were prepared with internal buffer of the following composition: 50 mM KCl, 10 mM NaCl, 60 mM KF, 2 mM MgCl2, 20 mM EGTA, 10 mM HEPES, pH 7.2., osmolarity: 288 mOsmol. Cells were prepared and patch clamped in an external buffer of the following composition: 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D-glucose monohydrate, 10 mM HEPES, pH 7.4, osmolarity: 298 mOsmol. 2.7. Statistical analyses The growth of cells was determined with single chemical treatment or multiple agents in combination, over time. Cell number over five days was analyzed for each treatment alone or in combination. The mean and SEM of treatment data points were then calculated. Treatment means were compared by using a one-way analysis of variance procedure (ANOVA), and a Student–Newman–Keul's multiple comparison test would follow if a significant treatment effect was observed. Concentration-response curves were constructed from growth measurements or voltage clamp currents, the latter taken at maximal current amplitude during a 500 msec activating pulse. Curves were generated by non-linear regression of log[agonist] vs. response or to a two-site binding model with GraphPad Prism™ (GraphPad Software, San Diego, CA, USA). Data were analyzed for each curve to determine the IC50 or EC50, 95% CI, and R2 (goodness of fit), as well as maximal effect of the treatment. 3. Results The known chloride channel blockers lindane and DIDS were tested against Sua1B cells to characterize any effects on cell growth (Fig. 1). A time-dependent response was demonstrated for Sua1B cells in the presence of varying concentrations of lindane (0.5–200 μM). No significant inhibition of cell number was observed on day 1 (Fig. 1A). Sua1B cell numbers were significantly inhibited at high concentrations of lindane, with 200 μM showing the highest amount of inhibition on days 2 (73%) and 3 (82%), with less inhibition of cell growth as concentrations decreased to 50 μM. No statistical significance was found in the response to lindane for lower concentrations (0.5–10 μM) on days 1–3 when compared to control (Fig. 1A). A concentration-response plot for lindane was run on Sua1B cell growth over a period of 3 days (Fig. 1B). Inhibition of cell growth after 1 day of exposure was modest and an IC50 value could not be reliably determined. The IC50 (CI) value for day 2
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Fig. 1. Effects of the organochlorine insecticide lindane on Sua1B insect cell number. (A) Time course curves for varying concentrations of lindane on Sua1B cells. (B) Concentrationresponse curves of lindane effects on Sua1B cell number over culture days 1–3. In B, the numbers of cells in solvent-treated controls are identical to those observed at the lowest concentration of lindane. In both plots, symbols are means ± SEM, and when error bars are absent, they are within the size of the symbol.
was 87 (44–172) μM, R2 = 0.84, and Hill slope of −0.67; and for day 3 was 68 (34–135) μM, R2 = 0.85, and a Hill slope = −0.72 (Fig. 1B). Sua1B cells displayed an increased Cl− current amplitude after the application of lindane (Fig. 2A,B), whereas DMSO (0.1%) showed no change. The effect of lindane varied with concentration, and was elevated significantly above basal levels at 100 μM (t-test, P b 0.05). Lindane stimulated chloride currents to a calculated maximum of 144% of control, with an EC50 of 116 (21–658) nM, R2 = 0.23, and a Hill slope of 1.6 (Fig. 2C). Lindane enhancement of chloride currents was blocked in extracellular solution without calcium ions (Fig. 2C). Current amplitudes in 1 mM lindane were lower than expected, and the current amplitude declined both with and without calcium. This effect may result from chloride current block by lindane at this concentration, and these data distorted the plot and curve fit, as reflected in the low R2 value (0.23). DIDS, a VSCC blocker [10], showed significant inhibition of cell growth at high concentrations, with 100 μM active on day 1 and declining in effect thereafter, while 200 μM was effective on all 3 days of incubation, when compared to control (Fig. 3A). No significant differences were observed on cell growth in the presence of 50 μM DIDS. A concentrationresponse plot for DIDS was run on Sua1B cell growth at day 2 of exposure, which showed an IC50 = 53 (48–80) μM, R2 = 0.93, and a Hill slope = 0.75 (Fig. 3B). The calculated minimum for the curve was 439 cells, for a maximal 46% inhibition of cell growth by DIDS. Because DIDS is a known blocker of Cl− currents and reduced the number of Sua1B cells, its effects on Cl− currents were studied using whole cell planar patch electrophysiological techniques. Cells displayed reduced current amplitude after the application of DIDS (100 nM1 mM), especially at positive potentials, and with increased inhibition at higher concentrations (10 μM-1 mM) (Fig. 4A). DMSO (0.1%)
controls showed no effect on the amplitude of the chloride current. A concentration-response plot for DIDS was run on Sua1B cells on day 2 of exposure, which showed an IC50 = 5.1 μM (0.15–174.5 μM), a Hill slope = −0.5, and an R2 = 0.63 (Fig. 4B). Sua1B cells only showed partial inhibition of Cl− current (69%) with DIDS even at high concentrations (1 mM). Some chloride currents (7 out of 19 patches) did not respond to DIDS inhibition, so recordings were performed in a low chloride buffer to confirm that they were chloride currents (Fig. 5A,B). In the presence of low chloride solution (35 mOsmol Cl− in low chloride saline solution compared to 297 mOsmol Cl− in regular external saline solution), DIDSinsensitive currents showed significantly reduced current amplitude, confirming that they were indeed chloride currents (Fig. 5A,B). No early inward currents consistent with fast sodium channels were observed in patch clamp studies. Chloride currents were also exposed to a pyrethroid of both the type 1 (allethrin) and type 2 (FNV) categories, respectively [11,12], and compared to control currents in DMSO (0.1%) to determine any significant effects on overall current amplitudes (Fig. 6). Control Cl− currents (Fig. 6A) were reduced about 27% by 10 μM allethrin (Fig. 6B), but substantially reduced by this same concentration of FNV (Fig. 6C). Replicated current–voltage relationships of different cells (n = 6) showed no significant reduction of current amplitudes in Sua1B cell recordings after application of 10 μM allethrin (Fig. 6D) when compared to control Cl− current amplitudes. In contrast, FNV (10 μM) displayed 60–70% reduction in current amplitude at this concentration when compared to control currents at −100 and +100 mV (Fig. 6E). In a previous study [13], we found that when applied to Sua1B cells alone, 1 μM VTD (a sodium channel activator) and TTX (a sodium
Fig. 2. Illustration of chloride channel currents under control (A) conditions and in the presence of 1 mM lindane (B). C. Concentration-dependence of increased chloride current in Sua1B cells when exposed to lindane (n = 9); extracellular solution containing calcium compared to the effect of lindane applied in extracellular solution without calcium (n = 6). Symbols are means ± SEM bars. Asterisks indicate a significant difference in lindane effects at a given concentration, in the presence and absence of calcium (unpaired T-test, P b 0.05).
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Fig. 3. Effects of DIDS on the number of Sua1B cells (A) at various incubation times (n = 3). Bars are means ± SEM. Statistical analysis was performed for each post-treatment day using one-way ANOVA (P b 0.05) and a Dunnett's multiple comparison post test. Letters represent statistical difference for a given day of exposure, and bars labeled by different letters are significantly different. Concentration-response curve of DIDS (B) on the number of Sua1B cells, corresponding to data for day 2 of the incubation (n = 3). Symbols are means ± SEM bars. When error bars are absent, they are within the size of the symbol.
channel blocker) showed no effect on cell growth. In the present investigation, when VTD was applied in combination with varying amounts of FNV, there was inhibition in cell number that was fit by a two-site binding model (Fig. 7). A potent effect of IC50 = 15 nM (4.5–49 nM), was found for the high affinity site and a lower potency effect with IC50 = 56 μM (25–124 μM) was found for the second site. The biphasic curve fit had an R2 = 0.951. Sua1B cells were then exposed to TTX over a 3 day period with combinations of VTD (1 μM) + FNV (1 μM or 100 μM). In these experiments, 1 μM TTX reversed the inhibitory effect of 0.1 μM FNV + 1 μM VTD on cell number, whereas high concentrations of FNV (100 μM + 1 μM VTD) showed inhibition of cell numbers that could not be reversed by TTX (Fig. 7, inset). 4. Discussion Two types of chloride channels previously identified in Sua1B cells [7] were characterized within this study. Outwardly rectifying voltage sensitive currents observed in native Sua1B cells seem to be mediated either primarily by CACCs or by a combination of CACCs and volumesensitive, outwardly-rectifying anion channels [7]. High concentrations of lindane, an organochlorine insecticide, inhibited growth of Sua1B cells, but the response did not occur in a typical concentration-dependent manner. Current amplitudes were increased at concentrations below 1 mM, which then reduced current amplitude (Fig. 2). The primary neurotoxic effects of lindane are usually attributed to its interference with γ-aminobutyric acid (GABA) receptor-ionophore complex; however, Abalis et al. [14] showed that lindane also binds with high affinity (Ki = 0.04 ± 0.01 μM) to VSCC in Torpedo electroplaque. Lindane, at the concentrations run in this study (50–200 μM), also affects intracellular calcium mobilization in rat myometrial smooth muscle cells (100–200 μM) [15], guinea pig macrophages (125 μM) [16] and
human HL-60 cells [17]. Such calcium mobilization could explain the effects of lindane at high concentrations on Sua1B cell growth, as well as activation of CACC (Fig. 3). However, any lindane-specific alterations in calcium ion homeostasis or signaling remain to be determined. Future studies utilizing calcium fluorescence assays in the presence and absence of lindane should be run to verify any possible effects on calcium ion flux/homeostasis. Outward Cl− currents expressed in Sua1B cells were mostly blocked by DIDS. The insecticidal/nematicidal properties of DIDS have been documented by Boina et al. [18,19], who observed paralysis in free living and plant parasitic nematodes, and was a stomach poison in European corn borer (Ostrinia nubilalis) larvae. Additional experiments on European corn borer disclosed that DIDS had a dose-dependent effect on larval midgut 36Cl− ion transport (IC50 = 7.4 μM) and midgut alkalinity (ca. IC50 = 29 μM) [19]. This latter action was similar to the potency found for DIDS on cell growth in this study (IC50 = 53 μM). Using the planar patch clamp technique, the inhibitory effect of DIDS on Cl− currents at +60 mV had IC50 = 5.1 μM, which is 10-fold greater potency than that observed in the cell growth studies, but similar to the IC50 for blocking 36Cl− uptake in European corn borer larval midgut. A similar potency of DIDS on Cl− currents was shown in rat peritoneal mast cells with IC50 = 2.3 μM and a Hill coefficient n = 0.7 [20]. Not all cells showed inhibition of current amplitude by DIDS, and this quality is shared by the cystic fibrosis transmembrane Cl− conductance regulator [21] and a certain ClC-3 type of chloride channel expressed in CHO cells [22]. The identity of the DIDS insensitive Cl− channel of Sua1B cells and the amino acids responsible for DIDS sensitivity in these channels remain to be determined. When characterizing the chloride channels present in Sua1B cells, this study found the majority of channels to be VSCCs, while those Diykov et al. [7] found were predominantly the CACC channel subtype.
Fig. 4. Electrophysiological effects of DIDS on chloride currents in Sua1B cells. (A) Current–voltage relationship of chloride current in Sua1B cells and inhibition with DIDS (n = 6). Symbols are means with error bars omitted for clarity. (B) Concentration-response curve representing percent inhibition of DIDS at +60 mV (n = 6). Symbols are means ± SEM bars.
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Fig. 5. DIDS insensitive chloride currents under planar patch conditions. Traces in (A) and (B) illustrate currents in normal (297 mOsmol) and low (35 mOsmol) chloride extracellular solutions, respectively. Plot (C) shows current–voltage relationship of DIDS-insensitive chloride channels in control and low chloride solutions (n = 3). Symbols are means ± SEM bars. Asterisks indicate a significant difference in current amplitude at a given voltage step in control or low chloride conditions (unpaired T-test, P b 0.05).
Both chloride channel subtypes were found in each study, but are thought to activate in different physiological circumstances and occur independently of one another. We do not know why the variation in chloride channel expression occurs, but it could be due to different aliquots/passages of Sua1B cells. Both studies showed the same reduction in current amplitude in the presence of DIDS (69%), confirming that a similar population of DIDS-sensitive chloride channels was present. Patch clamped Sua1B cells lack inward currents indicative of typical electrogenic sodium or calcium channels. However, in the presence of 1 μM VTD and increasing concentrations of FNV, cell death was increased in a dose-dependent, biphasic manner (Fig. 7). The high potency effect (EC50 = 15 nM) was completely inhibited in the presence of TTX, suggesting the possible presence of an “electrically-silent” sodium channel, similar to that previously described in rat C6 [23] and C9 cells [24]. In these cells, sodium currents cannot be activated by voltage clamp depolarization, but radiotracers can be used to measure chemical
activation of the channels by alkaloid or protein neurotoxins. Similar experiments could be used to characterize these putative sodium channels in Sua1B cells. The signs of intoxication by pyrethroids differ between the two categories (type 1 and 2) in mammals, where compounds described as type 1 (e.g., allethrin) display a tremor syndrome, whereas type 2 (e.g., FNV) present choreoathetosis (sinuous writhing) and salivation [25]. The different intoxication syndromes of the two classes of pyrethroid led to the hypothesis that there are other mechanisms involved in the toxicity of pyrethroids than just an action on sodium channels. Relevant to the present study is a proposed effect on voltage-gated chloride channels. Forshaw and Ray [26] showed a reduction of chloride conductance and an increase in membrane resistance in mammalian skeletal muscle, non-myelinated nerve fibers, and mouse neuroblastoma cells in the presence of a type 2 pyrethroid (deltamethrin), but not to a type 1 (cismethrin). In Sua1B cells, we observed an inhibition of
Fig. 6. Effects of pyrethroids on Sua1B cells using manual whole cell patch clamping technique. (A) shows control chloride current of a Sua1B cell before treatment. (B) and (C) are examples of chloride current inhibition by type 1 (allethrin) and type 2 (FNV) pyrethroids, respectively (10 μM each). Panels (D) and (E) show current–voltage relationships of control chloride currents and those currents in the presence of type 1 allethrin (n = 6, 10 μM) and type 2 FNV (n = 4, 10 μM) class of pyrethroids. Symbols are means + SEM and asterisks indicate a significant difference in current amplitude at a given voltage step (unpaired T-test, P b 0.05).
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[5]
[6]
[7] [8]
[9]
[10]
[11]
[12] Fig. 7. Inhibition of cell growth by FNV in the presence of a constant 1 μM concentration of VTD on cell number in Sua1B cells, and block of this effect by 1 μM TTX. Concentrationresponse curve of type 2 pyrethroid FNV taken on day 3 of incubation (n = 3 replicate experiments run on different days with different flasks of cells) is fit by a two site model. Symbols are means ± SEM bars. When error bars are absent, they are within the size of the symbol. Inset: TTX sensitivity of high potency (10−7 M FNV) and low potency (10−4 M FNV) effects, in separate experiments with matched controls (n = 3). Statistical analysis was performed using one-way ANOVA (P b 0.05) and a Student–Newman–Keul's multiple comparison post test. Bars labeled by different letters represents statistical significance (P b 0.05).
[13]
[14]
[15]
[16]
chloride current amplitude after application of 10 μM FNV (type 2), while no significant inhibition of the chloride current was shown in the presence of 10 μM allethrin (type 1). Such an effect of FNV on VSCC could contribute to the low potency inhibitory effect of FNV on Sua1B cell growth, but could also be due to mitochondrial effects, since Gassner et al. [27] showed pyrethroids are micromolar inhibitors of mitochondrial complex 1 in rat hepatocytes. To our knowledge, this finding is the first report of type 2 pyrethroid inhibition of insect VSCCs, and further research is required to explore any role it plays in pyrethroid intoxication and lethality.
[17]
Acknowledgments
[22]
This work was supported by USDA Specific Cooperative Agreement 58-0208-0-068 (to JRB) as part of the Deployed War Fighter Research Program. We would like to extend a special thanks to Dr. Michael Povelones for providing us with a stock of the Sua-1B cells.
[23]
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